1. Field of the Invention
The present invention relates generally to molecular biology and physiology. More specifically, the present invention relates to a novel regulatory element in the human LDL receptor promoter, designated FP1, which can be used to confer sterol regulatory capability to heterologous promoters and structural genes, or to screen for compounds capable of stimulating LDL receptor synthesis in a cell.
2. Description of the Related Art
Low density lipoprotein (LDL) receptor is a key component of the mechanism by which animal cells maintain balanced cholesterol homeostasis. The transcription of the LDL receptor gene is maintained under tight feedback regulation by cellular levels of sterols (Brown, M. S., and Goldstein, J. L., Science 232:34-47 (1986); and Goldstein, J. L., and Brown, M. S., Nature 343:425-30 (1990)). In cells with excess sterols, transcription is repressed; in contrast, transcription is accelerated in cells requiring cholesterol. This feedback regulation is most important in vivo in the liver. Ingestion of cholesterol in the diet decreases hepatic levels of the LDL receptor mRNA, and consequent decline in the hepatic LDL receptor causes LDL to accumulate in the circulation (Sorci-Thomas, M., et al., J. Biol. Chem. 264:9039-45 (1989); Mehta, K. D.,et al., J. Biol. Chem. 266: 10406-14 (1991); and Chang, R., et al., Biochem. Biophys. Res. Commun. 218:733-739 (1996)).
The target for sterol regulation lies within a stretch of 10 nucleotide base pairs that has been designated sterol regulatory element-1 (SRE-1) (Dawson, P. A., et al., J. Biol. Chem. 263:3372-79 (1988); and Smith, J. R., et al., J. Biol. Chem. 265:2306-10 (1995)). In addition to SRE-1, there are two Sp1-like sequences in the human LDL receptor gene promoter which bind to purified Sp1 (Mehta, K. D.,et al., J. Biol. Chem. 266:10406-14 (1991); Dawson, P. A., et al., J. Biol. Chem. 263:3372-79 (1988); and Kadonaga, J. T., et al., TIBS 11:20-23 (1986)). SRE-1s bind to SRE-1 binding proteins, which undergo proteolytic cleavage at the C-terminal membrane-associated domain (125 kDa) and are converted to functionally active nuclear forms (68 kDa) (Wang, X., et al, Cell 77:53-62 (1994); Sheng, Z., et al., Proc. Natl. Acad. Sci. USA 92:935-38 (1995); and Yokoyama, C., et al., Cell 75:187-97 (1994). The nuclear form of SRE-1 binding protein is transcriptionally active because it contains an acidic transcriptional activation domain and a basic helix-loop-helix leucine zipper region that mediates protein dimerization and DNA binding. All essential nucleotides of SRE-1 and Sp1 sites are conserved in evolution (Mehta, K. D.,et al., J. Biol. Chem. 266: 10406-14 (1991)).
In addition to regulation of the LDL receptor gene by cellular cholesterol levels, transcription is modulated by a variety of mitogenic and nonmitogenic signals in multiple cell types. Insulin and platelet-derived growth factor can stimulate LDL receptor gene transcription in quiescent mesenchymal cells (Mazzone, T., et al., J. Biol. Chem. 264:1787-92 (1989); Mazzone, T., et al., J. Biol. Chem. 265:5145-49 (1990); and Wade, D. P., et al., Eur. J. Biochem. 181:727-31 (1993)). Serum factors stimulate LDL receptor gene transcription in HepG2 cells, and mitogenic stimulation increases LDL receptor gene transcription in lymphocytes (Cuthbert, J. A., J. Lipid Res. 31:2067-78 (1990); and Ellsworth, J. L., et al., J. Cell Physiol. 135:213-23 (1988)). Additionally, cAMP, protein kinase C agonists, calcium ionophores, and arachidonic acid metabolites have been shown to affect LDL receptor expression in HepG2 cells (Auwerx, J. H., et al, Proc. Natl. Acad. Sci. USA 86:1133-37 (1989); Auwerx, J. H., et al., Mol. Cell. Biol. 9:2298-2302 (1989); and Krone, W., et al., J. Lipid Res. 29:1663-69 (1988)), and cytokines have been shown to modulate the LDL receptor pathway activity in endothelial cells, arterial smooth muscle cells, and HepG2 cells (Hamanaka, R., et al., J. Biol. Chem. 267:13160-65 (1992); Nicholson, A. C., and Hajjar, D. P., J. Biol. Chem. 267:25982-87 (1992); and Stopeck, A. T., et al., J. Biol. Chem. 268:17489-94 (1993)). Many of these stimuli increase the LDL receptor transcript, and some have been shown to enhance LDL receptor gene transcription. Induction of LDL receptor gene transcription by platelet-derived growth factor and insulin have been ascribed to the participation of Sp1 and SRE-1 sequences, respectively (Basheeruddin, K., et al., Arterioscler. Throm. Vasc. Biol. 15:1248-54 (1995); and Streicher, R., et al., J. Biol. Chem. 271:7128-33 (1996)). The mechanisms by which LDL receptor gene transcription respond to a variety of other humoral signals are not clearly understood.
The prior art is deficient in the lack of a novel regulatory element in the human LDL receptor promoter, designated FP1 used to confer sterol regulatory capability to previously known promoters and structural genes, and to screen for compounds capable of stimulating synthesis of LDL receptors in a cell. The present invention fulfills a long-standing need and desire in the art.